2.1. DNA-PROTEIN CROSSLINKING
DNA-protein interactions are important in almost every aspect of gene expression and inheritance (Stein, G. S. et al., eds., CHROMOSOMAL PROTEINS AND THEIR ROLE IN THE REGULATION OF GENE EXPRESSION, Academic Press, New York, 1979) and disruption of normal interactions has serious consequences (Costa, M., J. Cellular. Biochem. 44:127-135 (1990)). DNA-protein crosslinks have been shown to be induced by many established or suspected carcinogens such as UV and gamma radiation, BCNU (Banjar, Z. M. et al., Biochem. Biophys. Res. Commun. 114:767-773 (1983)), alkylating agents (Grunicke, H. et al., Cancer Res. 33:1048-1053 (1973)), formaldehyde (Cosma, G. N. et al., Mutat. Res. 201:161-168 (1988)) and some metal compounds such as nickel (Patierno, S. R. et al., Chem. Biol. Interactions 55:75-91 (1985)), chromate (Wedrychowski, A. et al., J. Biol. Chem. 260:7150-7155 (1985)), (Sugiyama, M. et al., Mol. Pharmacol. 29:606-613 (1986)) and cis- or trans-Pt(II) diammine dichloride (Banjar, Z. M. et al., Biochemistry 23:1921-1926 (1984)).
Nickel induces DNA-protein crosslinks indirectly via oxygen radicals (Salnikov, K. et al., Carcinogenesis 13:2341-2346 (1992)). Chromate can act directly by binding of the trivalent Cr to the phosphate backbone of DNA and linked to proteins through amino acid reactive groups such as the imidazole nitrogen of histidine or the hydroxyl group of tyrosine (Patierno, S. R. et al., supra).
The biological significance of DNA-protein crosslinks in vivo are still poorly understood. DNA-protein crosslinks are persistent in treated cells since their presence can be easily detected at long time intervals following removal of the crosslinking agent (Sugiyama, M. et al., supra; Tsapakos, M. J. et al., Cancer Res. 43:5662-5667 (1983)). Due to poor repair capacity, DNA-protein complexes may be present at critical phases of cellular function, e.g., during DNA replication, and thereby result in the loss of genetic material. This could, for example, inactivate tumor suppressor genes and lead to tumor formation. Because of the apparent low capacity for repair, the detection of this type of DNA damage should serve as a potentially important biological marker of exposure to various toxic agents.
2.2. DETECTION OF DNA-PROTEIN COMPLEXES
Many attempts have been made to develop methods of sufficient sensitivity and rapidity for detecting DNA-protein crosslinking occurring in cells exposed to crosslinking agents. The most widely used method for detecting and studying DNA-protein complexes is the alkaline elution method (Kohn, K. W. et al., DNA Repair: A Laboratory Manual of Research Procedures 1:378-401 Part B (1981)). The filter retention technique (Chiu, S.-M. et al., Int. J. Radiat Biol. 46:681-690 (1984)), as well as several other recently developed methods, have been utilized to detect the formation of DNA-protein complexes in intact cells (Cohen, M. D. et al., Anal. Biochem. 186:1-7 (1990); Miller, C. A. et al., Carcinogenesis 10:667-672 (1989); Chen, Y. et al., Carcinogenesis 12:1575-1580 (1991)). The alkaline elution method, though very sensitive, detects the lesions only indirectly, measuring the velocity at which DNA fragments pass through a filter. Protein-linked DNA passes more slowly than does free DNA (Kohn, K. W. et al., Biochem. Biophys. Acta 562:32-40 (1979)). Alkaline elution has a number of disadvantages. It is both time consuming and relatively cumbersome, allowing analysis of only a limited number of samples per assay. The technique does not allow isolation and characterization of DNA-protein complexes. Additionally, in many studies, the lysis stringency utilized has not been sufficient to dissociate all proteins bound non-covalently to DNA. For example, nickel (Ni) compounds induce formation of DNA-protein complexes in cultured cells and animal tissues as assessed by alkaline elution assay (Patierno, S. R. et al., supra); Cicarelli, R. B. et al., Cancer Res. 42:3544-3549 (1982)). However, using the alkaline elution assay, Ni-induced DNA-protein crosslinks detected with the detergent sarcosyl were disrupted by inclusion of the more stringent detergent, sodium dodecyl sulfate (SDS) at a 1% (v/v) concentration (Patierno, S. R. et al., Cancer Biochem. Biophys. 9:113-126 (1987)). Thus, under certain conditions, the results obtained using the alkaline elution method may be influenced by changes in chromatin conformation and/or composition. Therefore, the total DNA-protein complexes observed may not represent true covalent DNA-protein complexes.
A filter-binding technique has been used successfully to quantitate and biochemically characterize DNA-protein crosslinks (Oleinick, N. L. et al., Br. J. Cancer 55:135-140 (1987)). However, such measurement usually required high doses of crosslinking agents, and the dissociation conditions were similar to the alkaline elution procedure. In this system, the mechanism by which DNA-protein complexes bound to nitrocellulose filters remained obscure since free DNA did not bind while protein binding was only about 50%.
A number of other assays used to detect DNA-protein complexes (Wedrychowski, A. et al., J. Biol. Chem. 260:7150-7155 (1985); Patierno, et al., supra; Miller, C. A. et al., Mol. Carcinogenesis 1:125-133 (1988)) provided interesting data about the composition of DNA-protein complexes but also suffered from several disadvantages. They generally utilized ultracentrifugation steps requiring large amounts of starting material, were generally insensitive and were time-consuming.
An additional method for separating covalent DNA-protein complexes from non-covalent DNA-protein complexes was disclosed by Fisher et al., (PCT Publication WO90/04650, 3 May 1990). This method involves treating DNA-protein complexes with a reactive derivative of polyethylene glycol (PEG) to form covalent PEG-protein-DNA complexes which are then separated from DNA not bound to PEG by aqueous phase partition between an aqueous PEG phase and an aqueous phosphate phase. This method was said to allow the partitioning of DNA to which is bound a single protein molecule, such as topoisomerase, if the DNA is short enough. This method is said to be an improvement over the SDS-KCl method of Trask et al., (EMBO J. 3:671-676 (1984)) because the latter method is only efficient for sites where many topoisomerase molecules are attached to a single strand of DNA.
The K-SDS assay, above, was successfully used to characterize the covalent complexes formed between pure DNA (plasmid or virus) and topoisomerases in vitro. Sensitivity and reproducibility of this method depended upon DNA fragment size since the presence of a linked protein can precipitate different amounts of DNA depending upon its size. In the setting of pure DNA and protein, fragment size and background DNA-protein crosslinking were not a problem. However, use of a K-SDS assay to determine crosslinked DNA-protein in cell Freparations presents an additional set of hurdles which were overcome by the present invention.
Rowe, T. C. et al., Cancer Res. 46:2021-2026 (1986), adapted the K-SDS assay to quantitate DNA-topoisomerase complexes formed following exposure of L1210 cells to acridine derivatives. However, initial attempts by the present inventor to use the Rowe et al. modification of the K-SDS procedure to determine DNA-protein crosslinks in HOS and CHO cells were not successful because of the large amounts of precipitable DNA in untreated cells and the low reproducibility of the method. Additionally, the protocol disclosed for L1210 cells included the addition of salmon sperm DNA to ensure effective washing of the SDS pellet. This step precludes the measurement of non-radioactive (unlabeled) DNA for assessing crosslinking.
In general, the paucity of knowledge about the lesions which result from the covalent crosslinking of DNA with protein is due in part to the unavailability of a rapid, sensitive and simple method for detecting covalent DNA-protein complexes which could be used for early detection of damage in individuals exposed to metals through environmental contact. In particular, individuals occupationally exposed to metals, for example welders exposed to fumes containing various metal oxides and salts, would benefit from such a method. The present invention is directed to solving this problem.